Epithelial basement membrane is essential for the maintenance of epithelial integrity which is an important requirement for normal vision. To characterize the epithelial basement membrane of normal corneas and its alterations associated with different pathological conditions of the cornea, in vitro studies utilizing corneal epithelial cells in culture, and in vivo and in vitro studies utilizing immunochemical approaches will be carried out. We have successfully established normal rabbit and human corneal and conjunctival epithelial cells in culture using selective media containing D-valine, 5 or 6-fluorotryptophan and D-penicillamine. However, since the same media have not been suitable for long-term cultures of epithelial cells from dystrophic corneas, new media will be devised. Collagenous and noncollagenous glycoproteins secreted by these cells in culture will be characterized by direct biochemical analysis and by utilizing immunological approaches. Part of the normal or diseased corneal tissues will be utilized for immunochemical analysis of the epithelial basement membranes. Employing hybridoma technology, monoclonal antibodies will be developed to the components of basement membranes by using either tissue homogenate or partially purified basement membrane components of corneal or conjunctival epithelial cells as the immunogens. These antibodies will then be used to immunohistochemically study the distribution of the specific basement membrane antigens in normal and pathological corneas. These antibodies will also be utilized for immunoaffinity isolation of basement membrane components from cell cultures or from the tissues for further biochemical characterization. Some of the pathological conditions of the cornea and conjunctiva to be studies will include diabetes, keratoconus and ocular cicatritial pemphigoid. Immunological approaches will also be utilized to characterize the corneal reepithelialization process during wound healing in alkali burns or in penetrating wounds in rabbits. Monoclonal antibodies which recognize epithelial cells at various differentiation stages and basement membrane components will be utilized to monitor corneal and conjunctival epithelial cell migration and the synthesis of basement membrane components. Some of these antibodies have already been developed and partically characterized. Long-term objectives of these studies will be to understand the structure and function of epithelial basement membrane, corneal and conjunctival epithelial regeneration, and the differentiation processes in normal, diseased and injured tissues.